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Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Vasselon T, Hailman E, Thieringer R, Detmers PA - J. Exp. Med. (1999)

Bottom Line: Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized.These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14.In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.

ABSTRACT
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

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LPS aggregates formed in serum are internalized with mCD14–EGFP. U373–CD14–EGFP cells were incubated in HAP buffer with aggregated BODIPY–LPS (100 ng/ml) for 10 min at 37°C, washed, and incubated for an additional 30 min at 37°C before examination by confocal microscopy. The images for mCD14–EGFP fluorescence and BODIPY–LPS are shown in the top and center panels, respectively, and the merged image is in the bottom panel. There was colocalization of BODIPY–LPS and mCD14–EGFP on the cell surface and within many intracellular compartments.
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Figure 9: LPS aggregates formed in serum are internalized with mCD14–EGFP. U373–CD14–EGFP cells were incubated in HAP buffer with aggregated BODIPY–LPS (100 ng/ml) for 10 min at 37°C, washed, and incubated for an additional 30 min at 37°C before examination by confocal microscopy. The images for mCD14–EGFP fluorescence and BODIPY–LPS are shown in the top and center panels, respectively, and the merged image is in the bottom panel. There was colocalization of BODIPY–LPS and mCD14–EGFP on the cell surface and within many intracellular compartments.

Mentions: To determine whether mCD14 traffics with LPS aggregates during internalization, we incubated U373–CD14–EGFP with BODIPY–LPS aggregates at concentrations between 40 and 100 ng/ml and followed both BODIPY–LPS and mCD14–EGFP fluorescence by confocal microscopy. After incubation for 5–10 min at 37°C with BODIPY–LPS aggregates, the cells were washed and incubated further at 37°C. The aggregates bound to the cell surface and colocalized with mCD14–EGFP (Fig. 9). At least 15 min at 37°C was required to detect BODIPY–LPS in intracellular vesicles, suggesting that internalization of LPS aggregates was somewhat slower than internalization of LPS monomers in U373–CD14–EGFP. The mCD14–EGFP colocalized with BODIPY–LPS in intracellular vesicles detected at the earliest times, although not all of the vesicles that contained BODIPY–LPS also contained mCD14–EGFP. These results suggest that, in contrast with LPS monomers, LPS aggregates can remain bound to mCD14 during internalization. This supports the idea that there is more than one pathway for internalization of LPS.


Internalization of monomeric lipopolysaccharide occurs after transfer out of cell surface CD14.

Vasselon T, Hailman E, Thieringer R, Detmers PA - J. Exp. Med. (1999)

LPS aggregates formed in serum are internalized with mCD14–EGFP. U373–CD14–EGFP cells were incubated in HAP buffer with aggregated BODIPY–LPS (100 ng/ml) for 10 min at 37°C, washed, and incubated for an additional 30 min at 37°C before examination by confocal microscopy. The images for mCD14–EGFP fluorescence and BODIPY–LPS are shown in the top and center panels, respectively, and the merged image is in the bottom panel. There was colocalization of BODIPY–LPS and mCD14–EGFP on the cell surface and within many intracellular compartments.
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Related In: Results  -  Collection

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Figure 9: LPS aggregates formed in serum are internalized with mCD14–EGFP. U373–CD14–EGFP cells were incubated in HAP buffer with aggregated BODIPY–LPS (100 ng/ml) for 10 min at 37°C, washed, and incubated for an additional 30 min at 37°C before examination by confocal microscopy. The images for mCD14–EGFP fluorescence and BODIPY–LPS are shown in the top and center panels, respectively, and the merged image is in the bottom panel. There was colocalization of BODIPY–LPS and mCD14–EGFP on the cell surface and within many intracellular compartments.
Mentions: To determine whether mCD14 traffics with LPS aggregates during internalization, we incubated U373–CD14–EGFP with BODIPY–LPS aggregates at concentrations between 40 and 100 ng/ml and followed both BODIPY–LPS and mCD14–EGFP fluorescence by confocal microscopy. After incubation for 5–10 min at 37°C with BODIPY–LPS aggregates, the cells were washed and incubated further at 37°C. The aggregates bound to the cell surface and colocalized with mCD14–EGFP (Fig. 9). At least 15 min at 37°C was required to detect BODIPY–LPS in intracellular vesicles, suggesting that internalization of LPS aggregates was somewhat slower than internalization of LPS monomers in U373–CD14–EGFP. The mCD14–EGFP colocalized with BODIPY–LPS in intracellular vesicles detected at the earliest times, although not all of the vesicles that contained BODIPY–LPS also contained mCD14–EGFP. These results suggest that, in contrast with LPS monomers, LPS aggregates can remain bound to mCD14 during internalization. This supports the idea that there is more than one pathway for internalization of LPS.

Bottom Line: Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized.These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14.In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology and Chemical Biology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.

ABSTRACT
Lipopolysaccharide (LPS) fluorescently labeled with boron dipyrromethane (BODIPY) first binds to the plasma membrane of CD14-expressing cells and is subsequently internalized. Intracellular LPS appears in small vesicles near the cell surface and later in larger, punctate structures identified as the Golgi apparatus. To determine if membrane (m)CD14 directs the movement of LPS to the Golgi apparatus, an mCD14 chimera containing enhanced green fluorescent protein (mCD14-EGFP) was used to follow trafficking of mCD14 and BODIPY-LPS in stable transfectants. The chimera was expressed strongly on the cell surface and also in a Golgi complex-like structure. mCD14-EGFP was functional in mediating binding of and responses to LPS. BODIPY-LPS presented to the transfectants as complexes with soluble CD14 first colocalized with mCD14-EGFP on the cell surface. However, within 5-10 min, the BODIPY-LPS distributed to intracellular vesicles that did not contain mCD14-EGFP, indicating that mCD14 did not accompany LPS during endocytic movement. These results suggest that monomeric LPS is transferred out of mCD14 at the plasma membrane and traffics within the cell independently of mCD14. In contrast, aggregates of LPS were internalized in association with mCD14, suggesting that LPS clearance occurs via a pathway distinct from that which leads to signaling via monomeric LPS.

Show MeSH
Related in: MedlinePlus